At the request of the Environmental Protection Agency, the National Research Council (NRC) recently completed a major report entitled Toxicity Testing in the 21st Century: A Vision and a Strategy. The terms of reference for this report were to develop a long-range vision and strategic plan to advance the practices of toxicity testing and human health assessment of environmental agents. The report describes how current and anticipated scientific advances can be expected to transform toxicity testing to permit broader coverage of the universe of potentially toxic chemicals to which humans may be exposed, using more timely and more cost-effective methods for toxicity testing. The report envisages greatly expanded use of high- and medium-throughput in vitro screening assays, computational toxicology, and systems biology, along with other emerging high-content testing methodologies, such as functional genomics and transcriptomics. When fully implemented, the vision will transform the ways toxicity testing and chemical risk assessment are conducted, moving away from measuring apical health endpoints in experimental animals toward identification of significant perturbations of toxicity pathways using in vitro tests in human cells and cell lines. Population-based studies incorporating relevant biomarkers will also be useful in identifying pathway perturbations directly in humans and in interpreting the results of in vitro tests in the context of human health risk assessment. The present article summarizes and extends the NRC report and examines its implications for risk assessment practice.